A large variety of diseases, conditions, and disorders (hereinafter “indications”) are characterized as involving aberrantly proliferating cells. As used herein, “aberrantly proliferating cells” (or “aberrant cell proliferation”) means cell proliferation that deviates from the normal, proper, or expected course. For example, aberrant cell proliferation includes inappropriate proliferation of cells wherein DNA or other cellular components have become damaged or defective. Aberrant cell proliferation also characterizes clinical indications caused by, mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of cell death (e.g., apoptosis), or both. Such indications can be characterized, for example, by single or multiple local abnormal proliferations of cells, groups of cells or tissue(s), and include cancerous (benign or malignant) and noncancerous indications.
By definition, all cancers (benign and malignant) involve some form of aberrant cell proliferation. Some noncancerous indications also involve aberrant cell proliferation. Examples of noncancerous indications involving aberrant cell proliferation include rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus.
One approach to treating indications involving aberrantly proliferating cells involves the use of DNA damaging agents. These agents are designed to kill aberrantly proliferating cells by disrupting vital cellular processes such as DNA metabolism, DNA synthesis, DNA transcription, and microtubule spindle formation. They also can operate, for example, by introducing lesions into DNA that perturb chromosomal structural integrity. DNA damaging agents are designed and administered in ways that attempt to induce maximum damage and consequent cell death in aberrantly proliferating cells with a minimum damage to normal, healthy cells.
A large variety of DNA damaging agents has been developed to date, including chemotherapeutics and radiation, and others are in development. Unfortunately, the effectiveness of DNA damaging agents in treating conditions involving aberrant cell proliferation has been less than desired, particularly in the treatment of cancer. The selectivity of such agents for aberrantly proliferating cells over healthy cells (sometimes referred to as the therapeutic index) often is marginal.
Moreover, all cells have sensing and repair mechanisms that can work at cross purposes to DNA damaging agents. Such sensing mechanisms, called cell cycle checkpoints, help to maintain the order of the various cell replication stages and to ensure that each step is executed with high fidelity (Hartwell et al., Science, 246:629-634 (1989); Weinert et al., Genes Dev., 8:652 (1994)). When cells detect DNA damage, including damage purposefully induced by DNA damaging agents, certain signaling pathways activate cell cycle checkpoints and the cell replication cycle temporarily ceases (“arrests”). This arrest allows cells time to repair their DNA, often to a degree sufficient to allow them to continue to survive and proliferate. In the case of aberrantly proliferating cells, this repair is unwanted, as it may undermine efforts to induce DNA damage sufficient to kill such cells.
For example, the chemotherapeutic agent called GEMZAR™ (gemcitabine, or 2′,2′-difluoro-2′-deoxycytidine) damages DNA by incorporating itself into DNA during synthesis. Left unrepaired, damaged DNA generally is rendered incapable of sustaining life. In many targeted cells, however, cell cycle checkpoints detect the improperly made (or otherwise damaged) DNA. The activated cell cycle checkpoints trigger cell cycle arrest for a time sufficient to allow damaged DNA to be repaired. This is one way in which aberrantly proliferating cells are theorized to resist the cell-killing effect of DNA-damaging agents such as chemotherapeutics, radiation, and other therapies.
Other DNA-damaging agents cause tumor cells to arrest in S-phase. Tumor cells have been observed to resist certain chemotherapeutics simply by arresting in S phase while the chemotherapeutic agent is being administered. Then, as soon as the drug is removed, DNA damage is repaired, cell cycle arrest ceases, and the cells progress through the remainder of the cell cycle (Shi et al., Cancer Res. 61:1065-1072, 2001). Other therapeutics cause cell cycle arrest at other checkpoints, including G1 and G2. Inhibition of various DNA damage checkpoints therefore is expected to assist in preventing cells from repairing therapeutically induced DNA damage and to sensitize targeted cells to DNA damaging agents. Such sensitization is in turn expected to increase the therapeutic index of these therapies.
The cell cycle is structurally and functionally the same in its basic process and mode of regulation across all eukaryotic species. The mitotic (somatic) cell cycle consists of four phases: the G1 (gap) phase, the S (synthesis) phase, the G2 (gap) phase, and the M (mitosis) phase. The G1, S, and G2 phases are collectively referred to as interphase of the cell cycle. During the G1 phase, biosynthetic activities of the cell progress at a high rate. The S phase begins when DNA synthesis starts, and ends when the DNA content of the nucleus of the cell has been replicated and two identical sets of chromosomes are formed.
The cell then enters the G2 phase, which continues until mitosis starts. In mitosis, the chromosomes pair and separate, two new nuclei form, and cytokinesis occurs in which the cell splits into two daughter cells each receiving one nucleus containing one of the two sets of chromosomes. Cytokinesis terminates the M phase and marks the beginning of interphase of the next cell cycle. The sequence in which dell cycle events proceed is tightly regulated, such that the initiation of one cell cycle event is dependent on the completion of the prior cell cycle event. This allows fidelity in the duplication and segregation of genetic material from one generation of somatic cells to the next.
It has been reported that cell cycle checkpoints comprise at least three distinct classes of polypeptides, which act sequentially in response to cell cycle signals or defects in chromosomal mechanisms (Carr, Science, 271:314-315, 1996). The first class is a family of proteins that detect or sense DNA damage or abnormalities in the cell cycle. These sensors include Ataxia-telangiectasia Mutated protein (Atm) and Ataxia-Telangiectasia Rad-related protein (Atr). The second class of polypeptides amplify and transmit the signal detected by the detector and is exemplified by Rad53 (Alen et al. Genes Dev. 8:2416-2488, 1994) and Chk1. A third class of polypeptides includes cell cycle effectors, such as p53, that mediate a cellular response, for example, arrest of mitosis and apoptosis.
Much of the current understanding of the function of cell cycle checkpoints has been derived from the study of tumor derived cell lines. In many cases, tumor cells have lost key cell cycle check points (Hartwell et al., Science 266:1821-28, 1994). It has been reported that a key step in the evolution of cells to a neoplastic state is the acquisition of mutations that inactivate cell cycle checkpoint pathways, such as those involving p53 (Weinberg, Cell 81:323-330, 1995; Levine, Cell 88:3234-331, 1997). Loss of these cell cycle checkpoints results in the replication of tumor cells despite DNA damage.
Noncancerous tissue, which has intact cell cycle checkpoints, typically is insulated from temporary disruption of a single checkpoint pathway. Tumor cells, however, have defects in pathways controlling cell cycle progression such that the perturbation of additional checkpoints renders them particularly sensitive to DNA damaging agents. For example, tumor cells that contain mutant p53 are defective both in the G1 DNA damage checkpoint and in the ability to maintain the G2 DNA damage checkpoint (Bunz et al., Science, 282:1497-501, 1998). Checkpoint inhibitors that target initiation of the G2 checkpoint or the S phase checkpoint are expected to further cripple the ability of these tumor cells to repair DNA damage and, therefore, are candidates to enhance the therapeutic index of both radiation and systemic chemotherapy (Gesner, Abstract at SRI Conference: Protein Phosphorylation and Drug Discovery World Summit, March 2003).
In the presence of DNA damage or any impediment to DNA replication, the checkpoint proteins Atm and Atr initiate a signal transduction pathway leading to cell cycle arrest. Atm has been shown to play a role in a DNA damage checkpoint in response to ionizing radiation (IR). Atr is stimulated by agents that cause double strand DNA breaks, single strand DNA breaks, and agents that block DNA radiation.
Chk1 is a protein kinase that lies downstream from Atm and/or Atr in the DNA damage checkpoint signal transduction pathway (Sanchez et al., Science, 277:1497-1501, 1997; U.S. Pat. No. 6,218,109). In mammalian cells, Chk1 is phosphorylated in response to agents that cause DNA damage including ionizing radiation (IR), ultraviolet (UV) light, and hydroxyurea (Sanchez et al., supra; Lui et al., Genes Dev., 14:1448-1459, 2000). This phosphorylation which activates Chk1 in mammalian cells is dependent on Atm (Chen et al., Oncogene, 18:249-256, 1999) and Atr (Lui et al., supra). Furthermore, Chk1 has been shown to phosphorylate both wee1 (O'Connell et al., EMBO J., 16:545-554, 1997) and Pds1 (Sanchez et al., Science, 286:1166-1171, 1999), gene products known to be important in cell cycle control.
These studies demonstrate that mammalian Chk1 plays a role in the Atm dependent DNA damage checkpoint leading to arrest at S phase. A role for Chk1 in the S phase mammalian cells has recently been elucidated (Feijoo et al., J. Cell Biol., 154:913-923, 2001; Zhao et al., PNAS U.S.A, 99:14795-800, 2002; Xiao et al., J Biol Chem., 278(24):21767-21773, 2003; Sorensen et al., Cancer Cell, 3(3):247-58, 2003) highlighting the role of Chk1 in monitoring the integrity of DNA synthesis. Chk1 invokes an S-phase arrest by phosphorylating Cdc25A, which regulates cyclinA/cdk2 activity (Xiao et al., supra and Sorensen et al., supra). Chk1 also invokes a G2 arrest by phosphorylating and inactivating Cdc25C, the dual specificity phosphatase that normally dephosphorylates cyclin-B/cdc2 (also known as Cdk1) as cells progress from G2 into mitosis (Fernery et al., Science, 277:1495-7, 1997; Sanchez et al., supra; Matsuoka et al., Science, 282:1893-1897, 1998; and Blasina et al., Curr. Biol., 9:1-10, 1999). In both cases, regulation of Cdk activity induces a cell cycle arrest to prevent cells from entering mitosis in the presence of DNA damage or unreplicated DNA.
Additional classes of cell cycle checkpoint inhibitors operate at either the G1 or G2/M phase. UCN-01, or 7-hydroxystaurosporine, originally was isolated as a nonspecific kinase inhibitor having its primary effect on protein kinase C, but recently has been found to inhibit the activity of Chk1 and abrogate the G2 cell cycle checkpoint (Shi et al., supra). Thus, because UCN-01 is a nonselective Chk1 inhibitor, it is toxic to cells at high doses. At low doses, it nonspecifically inhibits many cellular kinases and also inhibits the G1 checkpoint (Tenzer et al., Curr. Med. Chem. AntiCancer Agents, 3:35-46, 2003).
UCN-01 has been used in conjunction with cancer therapies, such as radiation, the anticancer agent camptothecin (Tenzer et al., supra), and gemcitabine (Shi et al., supra), with limited success. In addition, UCN-01 has been used to potentiate the effects of temozolomide (TMZ) induced DNA mismatch repair (MMR) in glioblastoma cells (Hirose et al., Cancer Res., 61:5843-5849, 2001). In the clinic, UCN-01 is not an effective chemotherapeutic as expected, possibly due to a failure in treatment scheduling and a lack of identification of particular key molecular targets (Grant et al., Drug Resistance Updates, 6:15-26, 2003). Thus, Mack et al. report cell cycle-dependent potentiation of cisplatin by UCN-01 in a cultured nonsmall-cell lung carcinoma cell line, but do not identify with specificity the key cell cycle checkpoint(s) targeted by UCN-01. (Mack et al., Cancer Chemother. Pharmacol., 51(4):337-348, 2003).
Several other strategies exist for sensitizing tumor cells to treatment with cell cycle affecting chemotherapeutics. For example, administration of 2-aminopurine abrogates multiple cell cycle checkpoint mechanisms, such as mimosine-induced G1 arrest or hydroxyurea-induced S phase arrest, allowing the cell to progress into and through mitosis (Andreassen et al., Proc Natl Acad Sci U.S.A., 86:2272-2276, 1992). Caffeine, a methylxanthine, has also been used to enhance cytotoxicity of DNA-damaging agents, such as cisplatin and ionizing radiation, by mediating progression through the G2 checkpoint and thereby inducing cell death. (Bracey et al., Clin. Cancer Res., 3:1371-1381, 1997). However, the dose of caffeine used to accomplish the cell cycle abrogation exceeds clinically acceptable levels and is not a viable therapeutic option. Additionally, antisense nucleotides to Chk1 kinase have been used to increase sensitivity to the topoisomerase inhibitor BNP1350 (Yin et al., Biochem. Biophys. Res. Commun., 295:435-44, 2002), but demonstrate problems typically associated with antisense treatment and gene therapy.
Chk1 inhibitors have been disclosed, including aryl- and heteroaryl-substituted urea compounds described in U.S. patent application Ser. No. 10/087,715 and U.S. Provisional Patent Application Nos. 60/583,080, 60/585,292, and 60/602,968; diaryl urea compounds described in U.S. Patent Publication No. 2004/0014765, U.S. Patent Publication No. US2003/199511, U.S. Patent Publication No. 2004/0014765, and WO 03/101444; methylxanthines and related compounds described in Fan et al., Cancer Res. 55:1649-54. 1995; ureidothiphenes described in WO 03/029241 and WO 03/028731; N-pyrrolopyridinyl carboxamides described in WO 03/028724; antisense Chk1 oligonucleotides described in WO 01/57206 and U.S. Pat. No. 6,211,164; Chk1 receptor antagonists described in WO 00/16781; heteroaromatic carboxamide derivatives described in WO 03/037886; aminothiophenes described in WO 03/029242; (indazolyl)benzimidazoles described in WO 03/004488; benzimidazole quinolinones described in U.S. Patent Publication No. 20040092535 and WO 04/018419; heterocyclic-hydroxyimino-fluorenes described in WO 02/16326; scytoneman derivatives, such as scytonemin, described in U.S. Pat. No. 6,495,586; heteroarylbenzamides described in WO 01/53274; indazoles described in WO 01/53268; indolacarbazoles described in Tenzer et al., supra; chromane derivatives described in WO 02/070515; paullones described in Schultz et al., J. Med. Chem., Vol:2909-2919, 1999; indenopyrazoles described in WO 99/17769; flavones described in Sedlacek et al., Int J. Oncol., 9:1143-1168, 1996; peptide derivatives of peptide loop of serine threonine kinases described in WO 98/53050; oxindoles described in WO 03/051838; diazepinoindolones described in WO 2004/063198; pyrimidines described in WO 2004/048343; urea compounds described in WO 2004/014876; and pyrrolocarbazoles, benzofuroisoindoles, and azacyclopentafluorenes described in WO 2003/091255.
However, a need remains in the art for effective and selective inhibitors of Chk1. The present invention addresses this and other needs.